The objectives of this project are to determine some of the molecular mechanisms involved in the symbiotic interaction of Rhizobium and leguminous plants leading to biological nitrogen fixation. Our main approach will be to isolate and characterize, by the use of transposon-induced mutagenesis, molecular clonin and DNA sequencing, the Rhizobium genes responsible for the establishment of this symbiosis. This can be conveniently divided into 4 steps. (1) Specific transposon-induced mutants of Rhizobium, defective in symbiosis, will be identified by our rapid plant assay. (2) DNA extracted from these mutants will be cloned into E. coli plasmids and the clones carrying the transposon and flanking Rhizobium sequences selected by the antibiotic resistance carried on the transposon. (3) The cloned DNA fragments will be used as hybridisation probes to identify the corresponding wild-type genes contained in a cloned 'bank' of normal rhizobium DNA fragments. (4) Cloned DNA fragments carrying symbiotic genes will be sub-cloned into a conjugative plasmid and inserted back into the defective Rhizobium. Restoration of symbiotic capacity will then confirm the presence of a symbiotic gene. The availability of cloned symbiotic genes should enable us to characterize the genes involved by (a) direct DNA sequencing, (b) investigation of the effect of particular genes on various aspects of symbiosis such as the plant host range, and (c) comparison of the structure of particular genes in different Rhizobium species. While the above approach should allow us to isolate and characterize Rhizobium genes involved in each of the many steps leading to biological nitrogen fixation, including those coding for the nitrogenase enzyme complex itself, we also intend to isolate the genes involved in this latter step by a slightly different method. This will make use of the cross-hybridization between Rhizobium nif genes and sequences of the Klebsiella nif operon, as a means to identify specific clones carrying the Rhizobium nif sequences.